The present invention relates to a monoclonal antibody capable of specific binding to the 20 kDa molecular weight variant of human growth hormone, here called hGH 20K.
This monoclonal antibody has no substantial binding to the 22 kDa molecular weight variant hGH 22K.
It also relates to the use of this monoclonal antibody for measurement of hGH 20K, especially in body fluids.
The antibodies can be used for detection and quantification of hGH 20K, especially in serum.
The cell line has been deposited at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany, under deposit number DSM ACC 2254 on Feb. 28, 1996.
Human growth hormone (hGH) is a 22 kDa molecular weight single chain polypeptide (hGH 22K), composed of 191 amino acids with two intra-chain disulphide bonds, produced by the anterior pituitary gland (1,2). However, circulating hGH is a complex mixture of different molecular forms, some of which are pituitary-derived, such as hGH 22K and hGH 20K, a human growth hormone 20 kDa molecular weight single chain polypeptide, while others are secreted by the placenta during pregnancy (hGH-V). Furthermore, other tropic hormones, placental lactogen (hPL) and prolactin (PRL), show significant sequence identity with hGH. Other hGH molecular variants derived from post-translational modifications such as deamidation, acylation, glycosylation and oligomerization (3) have also been described.
Human growth hormone is coded for by two genes, hGH-N and hGH-V, which are clustered on chromosome 17 together with the highly homologous placental lactogen (hPL) gene (4,5). The main product of the pituitary-expressed hGH-N gene is the 191-amino-acid 22K hGH.
A secondary product of this gene is 20K hGH, derived by alternative mRNA splicing, which lacks 15 residues in the polypeptide chain, from amino acids 32 to 46 (6, 7). This hGH 20K represents 5-10% of pituitary hGH (3), and its biological properties have yet to be defined. While it certainly shares some functions of the 22K isoform, evidence also shows specific activities. Thus, hGH 20K does not bind to hGH receptors in human liver (8) or at least shows decreased binding (9) and has less insulin-like promoting activity (10). The 20K isoform competes with hGH for binding to rabbit mammary gland receptors, indicating that its effect is more like lactogen than somatogen, even though it promotes growth in the hypophysectomized rat (11) see also e.g. EP 587 427.
hGH 20K is cleared at a slower rate than hGH and this prolonged persistence of hGH 20K in the circulation may contribute to its higher than expected bioactivity in vivo (Baumann et al, Endocrinology, Vol 117, No 4, 1309-13, 1985).
In EP 587 427 a process for producing hGH 20K by a recombinant method is disclosed.
Despite the clinical relevance of this peptide hormone family, there is little information on the concentrations of circulating isoforms or the relative contribution of each molecular form of the complex mixture. Selective assays to define hGH 22K and hGH 20K concentration would be valuable both for diagnostics and basic research (12, 13, 14,). Tools such as monoclonal antibodies (mAb) which specifically block the effect of these proteins could be of great interest in helping to understand their specific biological actions.
It has been suggested that the amount and relation between hGH 22K and hGH 20K in circulation could be of importance for special diseases and state of illness, such as diabetes, acromegaly, chronical liver and/or renal disease, and a specific and precise method for measuring 20 kDa is thus highly needed.
No method, based on the use of a hGH 20K specific mAb, for specific detection and quantification exists today. Attempts have been made to raise hGH 20K specific mAbs with the purpose of developing immunoassays for hGH 20K, but without success. Reference can be given to F Gomez et al, J of Immnunoassay, 5 (364), 145-57 (1984). On page 155 it is stated xe2x80x9cSince the precise pathophysiological relevance of 20K GH is still largely unknown, we felt appropriate to develop an immunoassay for it, obtaining first monoclonal anti hGH 20K antibodies with high specificity. Nevertheless, no stable hybridoma secreting specific anti 20K antibodies could be obtained despite selective immunization of the animals with a highly purified preparation of the variantxe2x80x9d.
For a long time there has thus been a need for a hGH 20K antibodies with high specificity which could be used in a specific and precise method for measuring hGH 20K.
Such an antibody could possibly also be used for therapeutic applications involving blocking of the biological activity of hGH 20K.
We have now found a solution to the need of the detection and quantification of hGH 20K, as we have generated a mAb specific for hGH 20K.
This mAb has been successfully used to specifically detect this hormone in a variety of assays. This antibody has also been found to specifically block hGH 20K biological activity. For that reason it is useful to study the biological activity of this hormone and analyze its biological significance.
In specific examples we describe the generation and characterization of this mAb. As comparative antibodies, we also describe monoclonal antibodies specific for hGH 22K and one which recognizes both hGH 20K and hGH 22K.